Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying ®ve PDZ protein ± protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (K D ) of 8.1610 79 M. We ®nd that a naturally occurring splice insertion of ®ve amino acids (PDZ2b) abolishes its binding a nity to the APC protein. The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as b-catenin playing a major role in the regulation of cell division, migration and cell adhesion. Oncogene (2000) 19, 3894 ± 3901.

PTP-BL is a highly modular protein tyrosine phosphatase of unknown function. It consists of an N-terminal FERM domain, five PDZ domains, and a C-terminally located tyrosine phosphatase domain. Here we show that PTP-BL is involved in the regulation of cytokinesis. We demonstrate localization of endogenous PTP-BL at the centrosomes during inter- and metaphase and at the spindle midzone during anaphase. Finally PTP-BL is concentrated at the midbody in cytokinesis. We show that PTP-BL is targeted to the midbody and centrosome by a specific splicing variant of the N-terminus characterized by an insertion of 182 amino acids. Moreover, we demonstrate that the FERM domain of PTP-BL is associated with the contractile ring and can be cosedimented with filamentous actin, whereas the N-terminus can be cosedimented with microtubules. We demonstrate that elevating the expression level of wild-type PTP-BL or expression of PTP-BL with an inactive tyrosine phosphatase domain leads to defects in cytokinesis and to the generation of multinucleate cells. We suggest that PTP-BL plays a role in the regulation of cytokinesis.

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)-derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild-type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high-level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.

Semaphorins are a family of secreted and membraneassociated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/ PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses. Keywords: path ®nding, post-synaptic density, PSD-95, SAP90, semaphorin, synapse, synaptic plasticity.The semaphorins are a large family of secreted and membrane-bound proteins that are characterized by a conserved N-terminal located domain of < 500 amino acids, the sema domain (Kolodkin et al. 1993;Yu and Kolodkin 1999;Nakamura et al. 2000;Tamagnone and Comoglio 2000). According to their modular structure and membrane-binding capacity, semaphorins are divided into eight different classes (Semaphorin Nomenclature Committee 1999). Semaphorins play important roles in axon guidance, in the regulation of cell migration and angiogenesis (Behar et al. 1996;Kitsukawa et al. 1997), and in the modulation of the immune system (Hall et al. 1996;Delaire et al. 1998). Recent evidence shows additional functions of semaphorins in regulating neuronal apoptosis (Gagliardini and Fankhaus 1999;Shirvan et al. 1999) and metastatic growth of tumours (Yamada et al. 1997;Christensen et al. 1998). However, the function of semaphorins is best characterized as ensuring the correct connectivity of the nervous system during development (Mark et al. 1997). Only recently has there been major progress in identi®ying the signal transduction mechanisms of semaphorins. First, the neuropilins were identi®ed as transmembrane molecules binding class 3 semaphorins (Chen et al. 1997;He and Tessier-Lavigne 1997;Kolodkin et al. 1997). However, the cytoplasmic domain of neuropilin-1 is not necessary for Sema3A activity and consequently a second class of transmembrane semaphorin receptors was identi®ed, the plexins (Ohta et al. 1995;Comeau et al. 1998 Abbreviations used: DMEM, Dulbecco's modi®ed Eagle's medium; PBS, phosphate-buffered saline; PDZ, post-synaptic density protein-95, discs large protein, zonula occludens; PSD-95, post-synaptic density protein-95; SAP90, synapse associated protein 90; SDS2PA...

Metronidazole (Mtz) resistance in the gastric pathogen Helicobacter pylori is closely associated with inactivation of the nitroreductase gene rdxA. In order to identify respective mutations for diagnostic purposes we analyzed the rdxA gene in a collection of high-level Mtz-resistant clinical H. pylori isolates. Size alterations in the rdxA gene region were found in only two out of 45 and one out of 40 isolates showing lower-level (minimal inhibitory concentrations (MICs) 32-192 microg ml(-1)) and high-level (MIC>/=256 microg ml(-1)) Mtz resistance, respectively. Point mutations that interrupt the rdxA reading frame were detected in two out of eight high-level resistant isolates (MICs>/=256 microg ml(-1)). Most remarkably, the rdxA gene sequence was found to be identical in four out of five high-level Mtz-resistant and -susceptible paired H. pylori isolates from the same patients each. Taken together, these results demonstrate that although some isolates carry classical resistance-associated rdxA mutations, as described earlier, the use of rdxA mutations as a marker for prediction of Mtz resistance is limited.